Bacterial cellulose and bacterium producing it

ABSTRACT

[Problem] 
     To provide a bacterial cellulose which is highly dispersible in a liquid, shows excellent molding properties and high miscibility with other materials when applied to materials, and, therefore, has a high applicability as a practical material, and a bacterium which produces the bacterial cellulose. 
     [Solution] 
     A bacterial cellulose, water that contains said bacterial cellulose at a final concentration of 0.1±0.006 (w/w) showing a light transmittance at a wavelength of 500 nm of 35% or greater, and a bacterium producing the bacterial cellulose. According to the present invention, the bacterial cellulose that is uniformly dispersible in a liquid such as water can be obtained. The bacterial cellulose shows excellent molding properties and high miscibility with other materials and, therefore, can contribute to the improvement in the qualities of a final product or production efficiency thereof or to the reduction of production cost.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase Application of PCTInternational Application PCT/JP2013/085163, filed Dec. 27, 2013 whichclaims priority to Japanese Application No. 2012-289043, filed Dec. 28,2012, the contents of which are incorporated herein by reference intheir entireties for all purposes.

TECHNICAL FIELD

The present invention relates to a bacterial cellulose and a bacteriumproducing it, and particularly to a bacterial cellulose excellent indispersibility in liquids and a bacterium producing it.

BACKGROUND OF THE INVENTION

A bacterial cellulose typically consists of a nanofiber having a widthof about 50 nm, and has received attention as a material capable ofbeing utilized in various industrial fields since it hascharacteristics, such as high mechanical strength and biocompatibilityand biodegradability. The bacterial cellulose is typically obtained inthe form of a film consisting of a gelled substance (hereinafter,referred to as “gelled film”) on the culture medium surface bysubjecting a bacterium, such as an acetic acid bacterium, to stationaryculture; however, the gelled film has a problem, such as being poorlyapplicable as an actual material since it is poor in moldability andmiscibility with other substances when applied to materials and high incost because of being low in production efficiency.

To address such a problem, there is a need for a bacterial cellulose notin the form of a gelled film but dispersible in liquids and thereforeexcellent in applicability. For example, Non Patent Literature 1discloses a bacterial cellulose obtained by subjecting Acetobacterxylinum subsp. sucrofermentans to aerated and agitated culture, and NonPatent Literature 2 also discloses a bacterial cellulose obtained bysubjecting Gluconacetobacter xylinum strain JCM10150 to rotary shakingculture in a culture medium containing carboxymethyl cellulose (CMC).

CITATION LIST Non Patent Literature

-   Non Patent Literature 1: Yoshinaga, et al., Kagaku To Seibutsu    (Chemistry and Biology), vol. 35, no. 11, p. 7-14, 1997-   Non Patent Literature 2: S. Warashina, et al., 2010 Cellulose R&D    Abstracts at the 17th Annual Meeting of the Cellulose Society, p.    98, 2010

SUMMARY OF THE INVENTION Technical Problem

However, the bacterial cellulose described in Non Patent Literature 1 isnot high in dispersibility in water as is evident from the descriptionthat it is not one produced using a culture medium containing CMC havingthe effect of improving the dispersibility of a bacterial cellulose andthat it is dispersed “in the form of tiny grains or fibers” in water(ibid; page 9, right column). Consequently, the bacterial cellulose isinsufficient in terms of moldability and miscibility with othersubstances for practical use. The bacterial cellulose described in NonPatent Literature 2 is also not high in dispersibility in water sincewater containing the bacterial cellulose is higher in white turbidity atthe bottom than at the top and has sedimentation observed and cellulosegrains are visibly large (ibid; FIG. 1) in any of the cases where theamount of addition of CMC to the culture medium is 0.5%, 1%, and 2%.Consequently, this bacterial cellulose is insufficient in terms ofmoldability and miscibility with other substances, necessary forpractical use.

Thus, the bacterial celluloses described in both of Non PatentLiteratures 1 and 2 are insufficient in moldability as a material andmiscibility with other substances, and also poor in practicability interms of efficiency of material production.

The present invention has been made to solve such problems and an objectthereof is to provide a bacterial cellulose high in dispersibility inliquids, favorable in moldability and miscibility with other materialsin being put to practical use, and excellent in applicability as anactual material, and a bacterium producing the bacterial cellulose.

Solution to Problem

As a result of intensive studies, the present inventors have found thatthe bacterial cellulose is highly water-dispersible, which is obtainedby subjecting the strain SIID9587 as a new strain of Gluconacetobacterintermedius (accession number NITE BP-01495) (hereinafter, sometimesreferred to as “strain NEDO-01 (G. intermedius strain SIID9587)”) toagitated culture in a CMC-containing culture medium using aglycerol-containing by-product generated in producing a biodiesel fuelfrom vegetable oil (Bio Diesel Fuel By-product; BDF-B, waste glycerin),reagent glycerol, or molasses as a carbon source, thereby accomplishingthe following inventions.

(1) The bacterial cellulose according to the present invention has thephysical characteristic of a transmittance of light at a wavelength of500 nm of water containing the bacterial cellulose at a finalconcentration of 0.1±0.006% (w/w) of 35% or more.

(2) The bacterial cellulose according to the present invention furtherhas the physical characteristic of a retention volume of the peak top ofthe chromatogram in the gel permeation chromatography performed underthe following conditions i) to vi) of from 2.5 mL inclusive to 3.0 mLexclusive:

i) column: a column 6.0 mm in inside diameter and 15 cm in length,packed with a methacrylate polymer having a particle diameter of 9 μm;ii) guard column: 4.6 mm in inside diameter and 3.5 cm in length; iii)column temperature: 35° C.; iv) feed flow rate: 0.07 mL/minute; v)eluent: a 40 to 42% (w/w) tetrabutylphosphonium hydroxide aqueoussolution; and vi) final concentration of the bacterial cellulose in theeluent: 0.2% (w/w).

(3) The bacterial cellulose according to the present invention ispreferably produced by the assimilation of BDF-B.

(4) The bacterial cellulose according to the present invention ispreferably produced by the assimilation of 1 or 2 or more selected fromthe group consisting of sugar, a sucrose-containing by-product generatedin producing sugar, and hydrolysates thereof, and isomerized sugar.

(5) The by-product is preferably molasses when the bacterial celluloseaccording to the present invention is produced by the assimilation ofthe sucrose-containing by-product generated in producing sugar.

-   -   (6) The bacterial cellulose according to the present invention        may be one produced by Gluconacetobacter intermedius.

(7) The bacterial cellulose according to the present invention may beone produced by Gluconacetobacter intermedius strain SIID9587 (strainNEDO-01) (accession number NITE BP-01495).

(8) The bacterium according to the present invention is characterized byproducing the bacterial cellulose according to any one of (1) to (5)above.

(9) The bacterium according to the present invention may beGluconacetobacter intermedius strain SIID9587 (strain NEDO-01)(accession number NITE BP-01495) producing the bacterial celluloseaccording to any one of (1) to (5) above.

Advantageous Effects of Invention

The bacterial cellulose according to the present invention can provide abacterial cellulose almost uniformly dispersible in liquids such aswater, and can contribute to an improvement in the quality of the finalproduct and production efficiency or a reduction in production costsince this bacterial cellulose is excellent in moldability andmiscibility with other substances. The present invention can provide abacterial cellulose almost uniformly dispersible in liquids bypurification under mild conditions without requiring steps of refiningwith a mixer and the like, and can provide a bacterial cellulose havinga relatively large average molecular weight. In addition, the presentinvention can contribute to effective resource utilization by using asucrose-containing by-product generated in producing sugar, such asBDF-B or molasses, as a carbon source, and enables the achievement ofthe reduction of bacterial cellulose price. Further, the presentinvention can efficiently provide a large amount of a bacterialcellulose by production using Gluconacetobacter intermedius orGluconacetobacter intermedius strain SIID9587 (strain NEDO-01).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram showing a protocol for isolating a bacteriumproducing a bacterial cellulose by assimilating BDF-B. In the figure,the bacterial cellulose is abbreviated as BC. FIG. 2-1 is a diagramshowing points of identity and difference between the 16S rDNAnucleotide sequences of the strain SIID9587 and G. intermedius strainTF2. In the figure, the points of identity in the nucleotide sequencesare represented by *marks and the points of difference are representedby quadrangular boxes. In the figure, G. intermedius indicates G.intermedius strain TF2.

FIG. 2-2 is a diagram showing points of identity and difference betweenthe 16S rDNA nucleotide sequences of the strain SIID9587 and G.intermedius strain TF2. In the figure, the points of identity in thenucleotide sequences are represented by *marks and the points ofdifference are represented by quadrangular boxes. In the figure, G.intermedius indicates G. intermedius strain TF2.

FIG. 3 is a pair of tables showing bacteriological properties of thestrain SIID9587.

FIG. 4 is a series of charts showing IR spectra of a bacterial celluloseobtained by subjecting the strain NEDO-01 (G. intermedius strainSIID9587) to stationary culture (top chart) and products obtained byaerated and agitated culture using BDF-B and reagent glycerol as carbonsources (middle and bottom charts).

FIG. 5 is a series of photographs showing the appearance of waters eachcontaining bacterial celluloses obtained by subjecting the strainNEDO-01 (G. intermedius strain SIID9587) to aerated and agitated cultureand stationary culture (left and middle) and a pulp-derived bacterialcellulose nanofiber (right).

FIG. 6 is a series of drawings showing the light transmittance at awavelength of 500 nm of waters containing bacterial celluloses obtainedby subjecting strain NEDO-01 (G. intermedius strain SIID9587) to aeratedand agitated culture using molasses and reagent glycerol as carbonsources, respectively, and the amount of the bacterial celluloseproduced (amount of the BC produced) and the rate of production thereof(BC production rate).

FIG. 7 is a series of drawings showing the light transmittance at awavelength of 500 nm of waters containing bacterial celluloses obtainedby subjecting strain NEDO-01 (G. intermedius strain SIID9587) and theknown bacterial cellulose-producing bacteria G. hansenii strainATCC23769, G. xylinus strain ATCC53582, G. xylinus strain ATCC700178(BPR2001), G. xylinus strain JCM10150, G. intermedius strain DSM11804,and G. xylinus strain KCCM40274 to aerated and agitated culture, and theamount of the BC produced, the BC production rate, and the BC productionrate ratio.

FIG. 8 is a chart showing chromatograms of the gel permeationchromatography of a bacterial cellulose obtained by subjecting strainNEDO-01 (G. intermedius strain SIID9587) to rotation culture using BDF-Bas a carbon source (sample B), a pulp-derived cellulose nanofiber(pulp-derived CNF solution), and pullulan.

FIG. 9 is a pair of photographs showing the fiber widths and thetransmission electron microscope-observed images of bacterial cellulosesobtained by subjecting strain NEDO-01 (G. intermedius strain SIID9587)to aerated and agitated culture (agitated-culture BC solution) andstationary culture (mixer-treated stationary-culture BC solution).

FIG. 10 is a pair of photographs showing the transmission electronmicroscope-observed images of a bacterial cellulose obtained bysubjecting strain NEDO-01 (G. intermedius strain SIID9587) to aeratedand agitated culture (agitated-culture BC solution) and a pulp-derivedcellulose nanofiber (pulp-derived CNF solution).

FIG. 11 is a pair of photographs showing the polarizationmicroscope-observed images of a bacterial cellulose obtained bysubjecting strain NEDO-01 (G. intermedius strain SIID9587) to aeratedand agitated culture (agitated-culture BC solution) and a pulp-derivedcellulose nanofiber (pulp-derived CNF solution).

FIG. 12 is a graph showing the weight of bacterial celluloses obtainedby subjecting strain NEDO-01 (G. intermedius strain SIID9587) and theknown bacterial cellulose-producing bacteria G. hansenii strainATCC23769, G. xylinus strain ATCC53582, and G. xylinus strain ATCC700178(BPR2001) to stationary culture using reagent glycerol or BDF-B as acarbon source.

FIG. 13 is a graph showing the weight of bacterial celluloses obtainedby subjecting strain NEDO-01 (G. intermedius strain SIID9587) and theknown bacterial cellulose-producing bacteria, the strain ATCC53582 andthe strain ATCC23769, to shake culture using reagent glycerol or BDF-Bas a carbon source.

DETAILED DESCRIPTION OF THE INVENTION

The bacterial cellulose according to the present invention and abacterium producing it will be described below in detail. The bacterialcellulose according to the present invention refers to a celluloseproduced by a bacterium.

For the purpose of the present invention, bacterial cellulose “beingdispersed” in a liquid such as water refers to bacterial cellulose beingfloated or suspended in the liquid. The high dispersibility refers to,for example, the particle diameter or fiber width of a bacterialcellulose as a dispersoid being relatively small in a liquid, or thebacterial cellulose as a dispersoid being relatively uniformly floatedor suspended in the liquid.

The bacterial cellulose according to the present invention has a highdispersibility in such an extent that it is almost uniformly dispersedin a liquid. Here, the liquid in which the bacterial cellulose isdispersed may be any of an organic solvent and an aqueous solvent;however, an aqueous solvent is preferable.

How high or low the dispersibility of a bacterial cellulose is can bemeasured, for example, using the light transmittance as an index; therelationship holds true that higher dispersibility results in a largerlight transmittance and lower dispersibility results in a smaller lighttransmittance. The light transmittance can be determined by providingwater containing the bacterial cellulose at a predeterminedconcentration to a spectrophotometer, irradiating the water with lightat a predetermined wavelength, and measuring the amount of thetransmitted light.

The bacterial cellulose according to the present invention has thephysical characteristic of a transmittance of light at a wavelength of500 nm of water containing the bacterial cellulose at a finalconcentration of 0.1±0.006% (w/w) of 35% or more. Here, examples of thetransmittance of light at a wavelength of 500 nm of water containing thebacterial cellulose at a final concentration of 0.1±0.006% (w/w)according to the present invention can include 35% or more as well as36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 35% to99% (both inclusive), 36% to 99% (both inclusive), 37% to 99% (bothinclusive), 38% to 99% (both inclusive), 40% to 99% (both inclusive),35% to 95% (both inclusive), 36% to 95% (both inclusive), 37% to 95%(both inclusive), 38% to 95% (both inclusive), 40% to 95% (bothinclusive), 35% to 90% (both inclusive), 36% to 90% (both inclusive),37% to 90% (both inclusive), 38% to 90% (both inclusive), 40% to 90%(both inclusive), 35% to 85% (both inclusive), 36% to 85% (bothinclusive), 37% to 85% (both inclusive), 38% to 85% (both inclusive),40% to 85% (both inclusive), 35% to 80% (both inclusive), 36% to 80%(both inclusive), 37% to 80% (both inclusive), 38% to 80% (bothinclusive), and 40% to 80% (both inclusive).

The bacterial cellulose according to the present invention may also havea large average molecular weight compared to that of a plant-derivedcellulose, such as a pulp-derived cellulose nanofiber. The averagemolecular weight of a cellulose can be measured using, for example, achromatogram in the gel permeation chromatography as an index; therelationship holds true that a smaller molecular weight results in alarger retention volume of the peak top of such a chromatogram and alarger molecular weight results in a smaller retention volume.Specifically, the bacterial cellulose according to the present inventionmay have the physical characteristic of a retention volume of the peaktop of the chromatogram in the gel permeation chromatography performedunder the following conditions i) to vi) of from 2.5 mL inclusive to 3.0mL exclusive: i) the column is a column 6.0 mm in inside diameter and 15cm in length, packed with a methacrylate polymer having a particlediameter of 9 μm; ii) the guard column is 4.6 mm in inside diameter and3.5 cm in length; iii) the column temperature is 35° C.; iv) the feedflow rate is 0.07 mL/minute; v) the eluent is a 40 to 42% (w/w)tetrabutylphosphonium hydroxide aqueous solution; and vi) the finalconcentration of the bacterial cellulose in the eluent is 0.2% (w/w).

The bacterial cellulose according to the present invention can beproduced, for example, by causing a bacterium to produce a bacterialcellulose by culture in a culture medium containing a suitable carbonsource.

Here, examples of the carbon source can include monosaccharides, such asglucose and fructose; disaccharides, such as sucrose, maltose, andlactose; oligosaccharides; sugar; sucrose-containing by-productsgenerated in producing sugar, hydrolysates thereof, and isomerizedsugar; saccharides, such as starch hydrolysates; mannitol; ethanol;acetic acid; citric acid; glycerol; and BDF-B. The carbon source can beproperly set depending on the type of a bacterium, the cultureconditions, the cost of production, and the like. BDF-B consists of41.5% of glycerol, 21.4% of fatty acid, 12.4% of methanol, 6.3% ofignition residue, and 18.4% of others (Japan Food Research Laboratories)as a typical composition, and is a composition containing a large amountof glycerol available as a carbon source for a bacterium.

Here, sugar refers to a sweetener consisting essentially of sucrose(Kohjien, 6th Ed.), and, for the purpose of the present invention, maybe a chemically synthesized one, or one produced using a naturalproduct, such as sugar cane, sugar beet (white beet), sugar maple,gomuti (Borassus flabellifer), or sweet sorghum (Sorghum bicolordulciusculum), as a raw material. Examples of the sugar according to thepresent invention can include non-centrifugal sugar, such as muscovado,shiroshita-to, casonade (brown sugar), wasanbon, or maple sugar, andcentrifugal sugar, such as raw sugar or refined sugar. Examples of therefined sugar can include hard sugar, such as shirozara-to, coarsecrystal medium soft sugar, or granulated sugar; soft sugar, such aswhite superior soft sugar or yellow soft sugar; processed sugar, such ascube sugar, crystal sugar, powdered sugar, or frost sugar; and liquidsugar.

The sucrose-containing by-product generated in producing sugar refers toone containing sucrose among by-products generated in a step ofproducing sugar, and specific examples thereof can include the pomace ofnatural raw materials, such as sugar cane and sugar beet as abovedescribed; molasses; and the residue generated in a purification stepusing filtration or ion-exchange resin.

The hydrolysate of a disaccharide, an oligosaccharide, sugar, or asucrose-containing by-product generated in producing sugar refers to oneobtained by subjecting the disaccharide, oligosaccharide, sugar, orsucrose-containing by-product generated in producing sugar to hydrolysistreatment, such as heating in an acidic solution.

The components in the culture medium other than the carbon source may bethe same ones as those in well-known culture media used for the cultureof bacteria, and preferably contain CMC. Specific examples of such aculture medium can include common nutrient culture media containing CMC,nitrogen sources, inorganic salts, and, as needed, organic tracenutrients, such as amino acids and vitamins. Examples of the nitrogensource can include organic or inorganic nitrogen sources, such asammonium salts (e.g., ammonium sulfate, ammonium chloride, and ammoniumphosphate), nitrates, urea, or peptone. Examples of the inorganic saltcan also include phosphates, magnesium salts, calcium salts, iron salts,and manganese salts. Examples of the organic trace nutrient can includeamino acids, vitamins, fatty acids, nucleic acids, and further peptone,casamino acids, yeast extracts, and soybean protein hydrolysatescontaining the nutrients. When an auxotrophic mutant requiring aminoacids for growth is used, the required nutrients may further besupplemented.

The bacterium is not particularly limited provided that it can produce abacterial cellulose; however, preferred is a bacterium capable ofproducing the bacterial cellulose under agitated culture or aeratedculture, more preferably a bacterium assimilating BDF-B. Specificexamples thereof can include bacteria of the genus Acetobacter, thegenus Gluconacetobacter, the genus Pseudomonas, the genus Agrobacterium,the genus Rhizobium, and the genus Enterobacter. More specific examplesthereof can include Gluconacetobacter intermedius, Gluconacetobacterhansenii, Gluconacetobacter swingsii, Acetobacter pasteurianus,Acetobacter aceti, Acetobacter xylinum, Acetobacter xylinum subsp.sucrofermentans, Acetobacter xylinum subsp. nonacetoxidans, Acetobacterransens, Sarcina ventriculi, Bacterium xyloides, and Enterobacter sp.;however, among these, Gluconacetobacter intermedius is preferable. Stillmore specific examples thereof can include Gluconacetobacter intermediusstrain SIID9587 (strain NEDO-01) (accession number NITE BP-01495),Gluconacetobacter xylinus strain ATCC53582, Gluconacetobacter hanseniistrain ATCC23769, Gluconacetobacter xylinus strain ATCC700178 (BPR2001),Gluconacetobacter swingsii strain BPR3001E, Acetobacter xylinum strainJCM10150, and Enterobacter sp. strain CJF-002; among these,Gluconacetobacter intermedius strain SIID9587 (strain NEDO-01)(accession number NITE BP-01495) is preferable.

Culture methods can include, for example, agitated culture and aeratedculture. Specific examples of the agitated culture can include cultureusing a fermenter, not involving aeration (non-aerated and agitatedculture), culture using a fermenter, involving aeration (aerated andagitated culture), culture under swaying from side to side using abaffled flask (shake culture), and rotary culture using a baffled flask(rotation culture). The culture conditions may be well-known cultureconditions used for the culture of the above bacteria; examples thereofcan include culture conditions of an aeration volume of 1 to 10L/minute, a rotation number of 100 to 800 rpm, a temperature of 20 to40° C., and a culture period of 1 day to 7 days.

In the production of the bacterial cellulose according to the presentinvention, a step of pretreating a carbon source, a pre-preculture step,a preculture step, a step of purifying, drying, and suspending thebacterial cellulose, and the like may be carried out, as needed.

The bacterial cellulose according to the present invention can be used,for example, as an additive for paper strong agents, thickeners for foodproducts, suspension stabilizers, and the like.

Then, the bacterium according to the present invention produces theabove-described bacterial cellulose. For bacteria producing thebacterial cellulose according to the present invention, the same orequivalent components to those of the bacterial cellulose according tothe present invention will not be described again.

The bacterial cellulose according to the present invention and abacterium producing it will be described below based on Examples.However, the technical scope of the present invention is not intended tobe limited to the features exhibited by these Examples.

EXAMPLES Example 1 Isolation and Identification of Bacteria

(1) Isolation of Bacteria

Bacteria producing a bacterial cellulose by assimilating BDF-B wereisolated. Specifically, using the protocol shown in FIG. 1, enrichmentculture was first carried out employing a culture medium containing 2%(w/v) of reagent glycerol (a guaranteed reagent from Wako Pure ChemicalIndustries Ltd.) in place of glucose in Hestrin-Schramm standard culturemedium (composition; bacto pepton 0.5% (w/v), yeast extract 0.5% (w/v),Na₂HPO₄ 0.27% (w/v), citric acid 0.115% (w/v), glucose 2% (w/v); HSculture medium) (HS/glycerol culture medium) using apple and prune asseparation sources. The resultant bacteria were inoculated on anHS/glycerol culture medium containing a cellulose staining reagent andcultured on plates at 30° C., and 15 bacterial strains producingbacterial celluloses were selected. Subsequently, these strains wereinoculated on an LB culture medium (composition; trypsin 1% (w/v), yeastextract 0.5% (w/v), and sodium chloride 0.5% (w/v)) containing 2% (w/v)of reagent glycerol (a guaranteed reagent from Wako Pure ChemicalIndustries Ltd.) and subjected to stationary culture at 30° C. to formgelled films. The dry weight of the gelled films (hereinafter, referredto as “dry film weight”) was measured, and 8 strains for which the dryfilm weight was large were selected as bacteria assimilating glyceroland having a high bacterial cellulose-producing ability. Then, thesestrains were inoculated on an LB culture medium containing BDF-B andcultured on plates at 30° C., and further inoculated on the HS culturemedium and subjected to stationary culture at 30° C. to form gelledfilms. The operation of selecting a bacterial strain for which the dryfilm weight was large among these bacteria, culturing on plates with theglycerol-containing LB culture medium or the HS/glycerol culture medium,and then subjecting the resultant to stationary culture on the HSculture medium was repeated to select one bacterial strain having aBDF-B-assimilating property and having a high bacterialcellulose-producing ability, which was called strain SIID9587.

(2) Identification of Bacteria

Sequencing was carried out according to an ordinary method for thestrain SIID9587 of 1 (1) of this Example to determine the nucleotidesequence of the full-length 16S rDNA (1367 bp; SEQ ID NO: 1).Subsequently, 16S rDNA nucleotide sequence analysis and bacteriologicalproperty test were performed in TechnoSuruga Laboratory Co., Ltd.

[2-1] 16S rDNA Nucleotide Sequence Analysis

The 16S rDNA nucleotide sequence analysis was carried out using Aporon2.0 (TechnoSuruga Laboratory Co., Ltd.) as software and Aporon DB-BA 6.0(TechnoSuruga Laboratory Co., Ltd.) and the International NucleotideSequence Databases (GenBank/DDBJ/EMBL) as databases. As a result ofhomology search with Aporon DB-BA 6.0, the 16S rDNA nucleotide sequencefor the strain SIID9587 (SEQ ID NO: 1) was found to have high homologyto the 16S rDNA nucleotide sequence for the genus Gluconacetobacter andhave the highest homology to the 16S rDNA nucleotide sequence for G.intermedius strain TF2 (accession number Y14694) (homology rate: 99.8%).As a result of homology search with GenBank/DDBJ/EMBL, the 16S rDNAnucleotide sequence for the strain SIID9587 (SEQ ID NO: 1) was alsofound to have high homology to the 16S rDNA nucleotide sequence for thegenus Gluconacetobacter, and that for the type strain was found to havehigh homology to the 16S rDNA nucleotide sequence for G. intermediusstrain TF2 (accession number NR_(—)026435) (homology rate: 99.8%). Thesequence of the accession number Y14694 is identical to the sequence ofthe accession number NR_(—)026435. The results of the comparison betweenthe 16S rDNA nucleotide sequences for the strain SIID9587 and G.intermedius strain TF2 (accession number Y14694 or NR_(—)026435) areshown in FIGS. 2-1 and 2-2. As shown in FIGS. 2-1 and 2-2, 4 nucleotideswere different between both sequences. In homology search with AporonDB-BA 6.0, as a result of simplified molecular phylogenetic analysisbased on the 16S rDNA nucleotide sequences for the top 15 strains havinghigh homology, the strain SIID9587 was found to be included in thecluster formed by the species of the genus Gluconacetobacter.

[2-2] Bacteriological Property Test

The results of bacteriological property test are shown in FIG. 3. Asshown in FIG. 3, the strain SIID9587 was different in property in termsof not growing on a 5% acetic acid-containing culture medium from knownG. intermedius and not different in other properties therefrom (BRENNERet al., Bergey's manual of Systematic Bacteriology. Vol. 2. TheProteobacteria, Part C The Alpha-, Beta-, Delta-, andEpsilonproteobacteria. 2005. Springer. p72-77).

The above results of (2) [2-1] and [2-2] of this Example 1 showed thatthe strain SIID9587 belonged to Gluconacetobacter intermedius. On theother hand, it was shown that the strain SIID9587 was a new strain of G.intermedius since differences exist in the 16S rDNA nucleotide sequenceand the bacteriological property between the strain SIID9587 andGluconacetobacter intermedius strain TF2 as the type strain for G.intermedius as described above. Accordingly, this bacterial strain wasdeposited in the National Institute of Technology and Evaluation, PatentMicroorganisms Depositary (NITE-IPOD; #122, 2-5-8 Kazusakamatari,Kisarazu-shi, Chiba 292-0818, Japan) under the accession number NITEBP-01495, Dec. 21, 2012. Hereinafter, the Gluconacetobacter intermediusstrain SIID9587 (accession number NITE BP-01495) is called strainNEDO-01 (G. intermedius strain SIID9587).

(3) Determination of Product

The strain NEDO-01 (G. intermedius strain SIID9587) was precultured toproliferate bacterial cells. Subsequently, the culture solution obtainedby the preculture (preculture solution) was added to the HS culturemedium (carbon source; glucose), which was then subjected to stationaryculture at 30° C. for about 8 days to perform the main culture to form agelled film on the culture medium surface. The infrared spectroscopy(IR) spectrum and x-ray diffraction profile of the gelled film wereobtained and analyzed according to an ordinary method. As a result, thegelled film was shown to be a cellulose having a I-type crystalstructure. As a result of obtaining and analyzing a scanning electronmicroscope image according to an ordinary method, cellulose fibershaving a width of the nano order (cellulose nanofibers) were shown toform a network structure in the gelled film. From these results, thestrain NEDO-01 (G. intermedius strain SIID9587) was determined toproduce a cellulose.

Example 2 Evaluation of Product Obtained by Aerated and Agitated Culture

(1) Preparation of Product by Aerated and Agitated Culture

BDF-B was subjected to neutralization treatment and further subjected toautoclave treatment to provide pretreated BDF-B.

Culture media were prepared in which reagent glycerol (a guaranteedreagent from Wako Pure Chemical Industries Ltd.) was added in place ofglucose as a carbon source in an HS culture medium containing 2% (w/v)CMC (chemical grade, from Wako Pure Chemical Industries Ltd.) and inwhich the pretreated BDF-B was added to a concentration of 2% (w/v) inplace of glucose in the CMC-containing HS culture medium, and called amain-culture medium with glycerol and a main-culture medium with BDF-B,respectively. The strain NEDO-01 (G. intermedius strain SIID9587) wasfirst precultured to proliferate bacterial cells. Then, the preculturesolution was inoculated on 5 L each of the main-culture medium withglycerol and the main culture medium with BDF-B and using the fermenter,subjected to aerated and agitated culture for 4 days under conditions ofan aeration volume of 7 to 10 L/minute, a rotation number of 200 to 800rpm, and a temperature of 30° C. to perform main culture. A 1% (w/v)NaOH aqueous solution was added to the culture solution obtained by themain culture (main-culture solution), which was then shaken at 60° C.and 80 rpm for 4 to 5 hours to lyse bacterial cells. After subjectingthe resultant to centrifugation, the supernatant was removed to recoverthe precipitate to remove water-soluble bacterial cell components. Theoperation of adding ultrapure water thereto, performing centrifugation,and then removing the supernatant was repeated until the pH of theprecipitate in a wet state reaches 7 or less to purify the product, andthe resultant was called an agitated-culture BC solution.

(2) Preparation of Bacterial Cellulose by Stationary Culture

A gelled film was obtained by the method described in (3) of Example 1and cut to a size of about 1 cm×1 cm. Subsequently, a 1% (w/v) NaOHaqueous solution was added thereto, which was then shaken at 60° C. and80 strokes/minute for 4 to 5 hours and then shaken overnight at 20° C.The liquid was removed by filtration using a metal gauze to recover agelled film. The operation of adding ultrapure water thereto and shakingthe resultant overnight at 20° C. was repeated until pH reaches 7 orless for purification, followed by suspension treatment using a mixerfor several minutes, and the resultant was called a mixer-treatedstationary-culture BC solution.

(3) Analysis

The agitated-culture BC solution of (1) of this Example 2 and themixer-treated stationary-culture BC solution of (2) of this Example 2were each added dropwise onto a silicon plate, dried, and then providedto an infrared spectrophotometer (FT/IR-4200; JASCO Corporation), andmeasured at a cumulative number of 32 and a resolution of 2 cm⁻¹ or 4cm⁻¹ to provide an IR spectrum. The results are shown in FIG. 4. Asshown in FIG. 4, the IR spectra of the agitated-culture BC solutionsobtained using the main-culture medium with BDF-B and the main-culturemedium with glycerol had similar shapes to the IR spectrum of themixer-treated stationary-culture BC solution. From these results, theproduct obtained by subjecting the strain NEDO-01 (G. intermedius strainSIID9587) to agitated culture using BDF-B or reagent glycerol as acarbon source was determined to be a cellulose.

Example 3 Dispersibility of Bacterial Cellulose in Water

(1) Appearance of Water Containing Bacterial Cellulose

The agitated-culture BC solution using the main-culture solution withBDF-B of (1) of Example 2 and the mixer-treated stationary-culture BCsolution of (2) of Example 2 were provided. Commercial pulp-derivedcellulose nanofibers were added to water for dispersion, and theresultant was called a pulp-derived CNF solution. The agitated-cultureBC solution, the mixer-treated stationary-culture BC solution, and thepulp-derived CNF solution were allowed to stand for 1 day, followed byobserving their appearance. The results are shown in FIG. 5.

As shown in FIG. 5, the cellulose precipitation was observed in thepulp-derived CNF solution. Massive bacterial cellulose was observed inthe mixer-treated stationary-culture BC solution, showing that thedispersion state of the bacterial cellulose was non-uniform. Incontrast, in the agitated-culture BC solution, no precipitation ormassive bacterial cellulose was observed and the bacterial cellulose wasobserved to be in the state of being uniformly dispersed. These resultsshowed that the bacterial cellulose obtained by subjecting the strainNEDO-01 (G. intermedius strain SIID9587) to agitated culture had highdispersibility and was uniformly dispersed in a liquid, such as water,compared to the bacterial cellulose obtained by subjecting thepulp-derived cellulose nanofibers or the strain NEDO-01 (G. intermediusstrain SIID9587) to stationary culture.

(2) Light Transmittance of Water Containing Bacterial Cellulose

[2-1] Comparison Between Bacterial Cellulose Obtained by StationaryCulture and Pulp-Derived Cellulose

In the method described in (1) of Example 2, rotation culture wasperformed under conditions of 150 rpm and a temperature of 30° C. for 3days using a baffled flask in place of the fermenter as main culture toprepare agitated-culture BC solutions, which were called sample A(obtained using the main-culture medium with glycerol) and sample B(obtained using the main-culture medium with BDF-B). Theagitated-culture BC solution obtained using the Main-culture medium withBDF-B of (1) of Example 2 was called sample C, and the agitated-cultureBC solution obtained using the main-culture medium with glycerol wascalled sample D. The mixer-treated stationary-culture BC solution of (2)of Example 2 and the pulp-derived CNF solution of (1) of Example 3 wereprovided. These solutions were adjusted to a final celluloseconcentration of 0.1±0.006% (w/w) and 1 mL each thereof were added tocells and subjected to a spectrophotometer (U-2001 double-beamspectrophotometer; Hitachi, Ltd.) to measure the transmittance of lightat a wavelength of 500 nm. A polyethylene disposable cuvette(semi-micro, having a light path length of 10 mm and a light path widthof 4 mm) was used as each cell, and ultrapure water was used as areference. The results are shown in Table 1.

TABLE 1 Final Concentration of Cellulose Culture Method Carbon Source (%(w/w)) Transmittance (%) Sample A Agitated culture (Baffled Flask)Reagent Glycerol 0.10505 74.75 Sample B Agitated culture (Baffled Flask)BDF-B 0.10309 70.53 Sample C Agitated culture (Fermenter) BDF-B 0.0957063.82 Sample D Agitated culture (Fermenter) Reagent Glycerol 0.1037549.66 Mixer-Treated Stationary- Stationary culture Glucose 0.09964 19.19culture BC Solution Pulp-Derived CNF Solution 0.10514 12.72

As shown in Table 1, the transmittance of the samples A, B, C, and D was74.75%, 70.53%, 63.82%, and 49.66%, respectively, prominently highcompared to 19.19% for the mixer-treated stationary-culture BC solutionand 12.72% for the pulp-derived CNF solution, and roughly in the rangeof from 40% to 80% (both inclusive).

[2-2] Comparison Between Presence and Absence of CMC in Culture Medium

In the method described in (1) of Example 2, the HS culture mediumcontaining 2% (w/v) CMC and the HS culture medium containing no CMC wereeach used to provide agitated-culture BC solutions. However, molasseswas used in place of glucose as a carbon source. When molasses was usedas a carbon source, the number of days in the main culture was set to 3days in place of 4 days since the carbon source in the culture mediumvirtually disappeared at day 3 of the main culture. Subsequently, thelight transmittance of bacterial cellulose-containing waters wasmeasured by the method described in (2) [2-1] of Example 3. The resultsare shown in the following Table 2.

TABLE 2 CMC in Carbon Transmittance Culture Medium Culture Method Source(%) Contain Agitated culture Molasses 57 Not Contain Agitated cultureMolasses 18

As shown in Table 2, the transmittance when the HS culture mediumcontaining CMC was used was 57%, whereas the transmittance when the HSculture medium containing no CMC was used was 18%.

The above results of (2) [2-1] and [2-2] of this Example 3 showed thatthe water containing the bacterial cellulose obtained by subjecting thestrain NEDO-01 (G. intermedius strain SIID9587) to agitated culture inthe CMC-containing culture medium at a final concentration of 0.1±0.006%(w/w) had a transmittance of light at a wavelength of 500 nm of 40% to80% (both inclusive). In other words, the agitated culture of the strainNEDO-01 (G. intermedius strain SIID9587) in the CMC-containing culturemedium was shown to provide a bacterial cellulose having a prominentlyhigh dispersibility in a liquid and uniformly dispersible in the liquid.

Example 4 Comparison in Transmittance and Bacterial Cellulose ProductionRate Between Different Carbon Sources

Agitated-culture BC solutions were each obtained by the method describedin (1) of Example 2. However, molasses and reagent glycerol were used ascarbon sources in place of glucose. When molasses was used as a carbonsource, the number of days in the main culture was set to 3 days inplace of 4 days. Subsequently, the light transmittance of each bacterialcellulose-containing water was measured by the method described in (2)[2-1] of Example 3. The agitated-culture BC solution was dried tomeasure the absolute dry weight of the bacterial cellulose, and theconcentration of the bacterial cellulose per 1 L of the culture mediumwas calculated based on the measurement results and defined as theamount of the bacterial cellulose produced (amount of BC produced; g/L).A value provided by dividing the amount of BC produced by the number ofdays in the main culture is calculated, and the value was defined as thebacterial cellulose production rate (BC production rate; g/L/day). Theresults are shown in FIG. 6.

As shown in the table and left bar graph of FIG. 6, the transmittancewhen molasses was used as a carbon source was 57% and was the same (57%)as that when reagent glycerol was used as a carbon source. These resultsshowed that the culture of the strain NEDO-01 (G. intermedius strainSIID9587) using molasses as a carbon source provided a bacterialcellulose having a high light transmittance at a wavelength of 500 nm ofwater containing the bacterial cellulose at a final concentration of0.1±0.006% (w/w) and was the same as when reagent glycerol was used as acarbon source. In other words, the culture of the strain NEDO-01 (G.intermedius strain SIID9587) using molasses as a carbon source was shownto provide a bacterial cellulose having high dispersibility anduniformly dispersible in a liquid.

As shown in the table and right bar graph of FIG. 6, the BC productionrate when molasses was used as a carbon source was 1.48 g/L/day and wasabout 1.5 times higher than that (0.95 g/L/day) when reagent glycerolwas used as a carbon source. These results showed that the culture ofthe strain NEDO-01 (G. intermedius strain SIID9587) using molasses as acarbon source provided a bacterial cellulose having high dispersibilityin high amounts in a short period of time.

Example 5 Comparison in Transmittance and Bacterial Cellulose ProductionRate Between Different Bacteria

An agitated-culture BC solution was obtained by the method described in(1) of Example 2. However, molasses was used as a carbon source in placeof glucose. The strain NEDO-01 (G. intermedius strain SIID9587) andGluconacetobacter hansenii strain ATCC23769, Gluconacetobacter xylinusstrain ATCC53582, Gluconacetobacter xylinus strain ATCC700178 (BPR2001),Gluconacetobacter xylinus strain JCM10150, Gluconacetobacter intermediusstrain DSM11804, and Gluconacetobacter xylinus strain KCCM40274 as knownbacterial cellulose-producing bacteria were used as bacteria,respectively. When the strain NEDO-01 (G. intermedius strain SIID9587)was used, the number of days in the main culture was set to 3 days inplace of 4 days since the carbon source in the culture medium virtuallydisappeared at day 3 of the main culture. On the other hand, when thestrain DSM11804 was used, the number of days in the main culture was setto 5 days in place of 4 days since the decrease in the carbon source inthe culture medium was small in magnitude even at day 4 of the mainculture. Subsequently, the light transmittance of each bacterialcellulose-containing water was measured by the method described in (2)[2-1] of Example 3. The amount of BC produced (g/L) and the BCproduction rate (g/L/day) were calculated by the method described inExample 4, and the transmittance and the BC production rate werequantified in bar graphs. The results are shown in FIG. 7.

As shown in the table and left bar graph of FIG. 7, the transmittancewhen the strain NEDO-01 (G. intermedius strain SIID9587) was used was57%, whereas the transmittance when G. hansenii strain ATCC23769, G.xylinus strain ATCC53582, G. xylinus strain ATCC700178 (BPR2001), G.xylinus strain JCM10150, G. intermedius strain DSM11804, and G. xylinusstrain KCCM40274 were used was 20%, 33%, 29%, 27%, 9%, and 13%,respectively. These results showed that the transmittance of light at awavelength of 500 nm of the water containing the bacterial celluloseobtained by culturing the strain NEDO-01 (G. intermedius strainSIID9587) at a final concentration of 0.1±0.006% (w/w) was prominentlyhigh (35% or more) compared to the light transmittance of the watercontaining the bacterial cellulose obtained by culturing each of thestrains other than NEDO-Ol (G. intermedius strain SIID9587). In otherwords, the culture of the strain NEDO-01 (G. intermedius strainSIID9587) was shown to be capable of providing a bacterial cellulosehaving high dispersibility and uniformly dispersible in a liquid.

As shown in the table and right bar graph of FIG. 6, the BC productionrate when the strain NEDO-01 (G. intermedius strain SIID9587) was usedwas 1.48 g/L/day, whereas the BC production rate when G. hansenii strainATCC23769, G. xylinus strain ATCC53582, G. xylinus strain ATCC700178(BPR2001), G. xylinus strain JCM10150, G. intermedius strain DSM11804,and G. xylinus strain KCCM40274 were used was 1.05 g/L/day, 1.03g/L/day, 1.11 g/L/day, 1.10 g/L/day, 0.42 g/L/day, and 0.43 g/L/day,respectively. In other words, the BC production rate when the strainNEDO-01 (G. intermedius strain SIID9587) was used was prominently highcompared to the BC production rate when the strains other than NEDO-01(G. intermedius strain SIID9587) were used. These results showed thatthe culture of the strain NEDO-01 (G. intermedius strain SIID9587) couldprovide a bacterial cellulose having high dispersibility in high amountsin a short period of time.

Example 6 Molecular Weight of Bacterial Cellulose

The samples A, B, C, and D and pulp-derived CNF solution of (2) ofExample 3 were provided as samples. These samples were eachfreeze-dried, added to a 57 to 59% tetrabutylphosphonium hydroxideaqueous solution, and dissolved by standing at 35° C., followed byadding water to a tetrabutylphosphonium hydroxide concentration of 40 to42% (w/w) and a sample concentration of 0.2% (w/w). Subsequently,centrifugation was carried out to precipitate impurities to recover thesupernatant. The supernatant was subjected to the gel permeationchromatography under the following conditions to measure the retentionvolume of the peak top of the chromatogram. The supernatant was measured3 times under the same conditions. The results are shown in Table 3, anda randomly selected chromatogram is shown in FIG. 8.

Condition for Gel Permeation Chromatography

Instrument; high-performance liquid chromatograph (Shimadzu Corporation)

Column; a column 6.0 mm in inside diameter and 15 cm in length, packedwith a methacrylate polymer having a particle diameter of 9 μm (TSKgelsuper AWM-H; Tosoh Corporation) Guard column; 4.6 mm in inside diameterand 3.5 cm in length (TSK guardcolum super AW-H; Tosoh Corporation)

Column temperature; 35° C.

Feed flow rate; 0.07 mL/minute

Sample injection volume; 10 μL

Eluent; a 40 to 42% (w/w) tetrabutylphosphonium hydroxide aqueoussolution

Final concentration of bacterial cellulose in the eluent; 0.2% (w/w)

Control sample; pullulan having a molecular weight of 85.3×10⁴ (Shodexstandard P-82)

TABLE 3 Standard Retention Retention Average/ Deviation/ Time/MinuteVolume/mL mL mL Sample A (1st) 40.4 2.828 2.79 0.05 Sample A (2nd) 39.12.737 Sample A (3rd) 40 2.8 Sample B (1st) 39.8 2.786 2.81 0.03 Sample B(2nd) 39.9 2.793 sample B (3rd) 40.7 2.849 Sample C (1st) 40.1 2.8072.82 0.02 Sample C (2nd) 40.5 2.835 Sample C (3rd) 40.1 2.807 Sample D(1st) 39.2 2.744 2.76 0.02 Sample D (2nd) 39.4 2.758 Sample D (3rd) 39.82.786 Pulp-derived 42.9 3.003 3.04 0.04 CNF Solution (1st) Pulp-derived43.4 3.038 CNF Solution (2nd) Pulp-derived 43.9 3.073 CNF Solution (3rd)Pullulan (1st) 45.7 3.199 3.24 0.04 Pullulan (2nd) 46.8 3.276 Pullulan(3rd) 46.4 3.248

As shown in Table 3 and FIG. 8, the retention volume of the peak top ofeach of the samples A, B, C, and D was on average 2.79 mL, 2.81 mL, 2.82mL, and 2.76 mL, respectively and small compared to 3.04 mL for thepulp-derived CNF solution and 3.24 mL for pullulan. These results showedthat the average molecular weight of the bacterial cellulose obtained bysubjecting the strain NEDO-01 (G. intermedius strain SIID9587) toagitated culture was larger than that of the pulp-derived cellulose andmore than 85.3×10⁴ in terms of pullulan. Table 3 also showed that whenthe bacterial cellulose obtained by subjecting the strain NEDO-01 (G.intermedius strain SIID9587) to agitated culture was subjected to thegel permeation chromatography under the above conditions, the retentionvolume of the peak top of the chromatogram reached 2.5 mL (inclusive) to3.0 mL (exclusive) since the retention volume of the peak top of each ofthe samples A, B, C, and D was in the range of 2.737 to 2.849 mL.

Example 7 Morphology of Bacterial Cellulose

(1) Measurement of Fiber Width

The agitated-culture BC solution using the main-culture medium withglycerol of (1) of Example 2 and the mixer-treated stationary-culture BCsolution of (2) of Example 2 were provided. These cellulose solutionswere each adjusted to a concentration of about 0.001% (w/w), and then,10 μL of each solution was added dropwise onto a Formvar-coated coppergrid and air-dried. Subsequently, 5 μL of a 5% (w/v) gadolinium acetateaqueous solution was added dropwise thereto, and the excess solution wasremoved with a paper filter 10 seconds later for negative staining. Theresultant was observed under a transmission electron microscope at anacceleration voltage of 80 kV and an observation magnification of 30,000times to measure the width of cellulose fibers based on the observedimage. The results are shown in FIG. 9.

As shown in FIG. 9, the width of the cellulose fibers was 17±8 nm forthe agitated-culture BC solution, was prominently small compared to55±22 nm for the mixer-treated stationary-culture BC solution, and had asmall standard deviation. These results showed that the bacterialcellulose obtained by subjecting the strain NEDO-01 (G. intermediusstrain SIID9587) to agitated culture formed fine and uniform fibersshowing small variations in width between the fibers.

(2) Determination of Uniformity of Fiber Width and Aggregation State

The agitated-culture BC solution using the main-culture medium withBDF-B of (1) of Example 2 and the pulp-derived CNF solution of (1) ofExample 3 were provided. These cellulose solutions were each adjusted toa concentration of about 0.01% (w/w), and then, the operation ofspraying the solution on a Formvar-coated copper grid and drying itusing a dryer was repeated 10 times. Subsequently, 5 μL of a 5% (w/v)gadolinium acetate aqueous solution was added dropwise thereto, and theexcess solution was removed with a paper filter. In addition, thesequence of dropwise adding 5 μL of ultrapure water and then removingthe excess solution with a paper filter was repeated 2 times, followedby negative staining by air-drying. The resultant was observed under atransmission electron microscope at an acceleration voltage of 80 kV andan observation magnification of 10,000 times. The results are shown inFIG. 10. It was also observed with crossed nicols using a polarizingmicroscope. The results are shown in FIG. 11.

As shown in FIG. 10, many cellulose fibers having comparable widths ofthe nano-scale were observed in the agitated-culture BC solution,whereas cellulose fibers having various widths, including widths aslarge as about 500 nm or more, were observed in the pulp-derived CNFsolution. From these results, it was again determined that the bacterialcellulose obtained by subjecting the strain NEDO-01 (G. intermediusstrain SIID9587) to agitated culture formed fibers having a uniformwidth of the nano-scale.

As shown in FIG. 11, relatively thick fibers as shown by arrows weredefinitely observed in the pulp-derived CNF solution, whereas dim imageswere observed in the portion enclosed by a dotted line in theagitated-culture BC solution. These results showed that relatively thickfibers, such as submicrofibers and microfibers, were present for thepulp-derived cellulose, whereas thin fibers of the nano-scale wereuniformly dispersed for the bacterial cellulose obtained by subjectingthe strain NEDO-01 (G. intermedius strain SIID9587) to agitated culture.

Example 8 Evaluation of Bacterial Cellulose-Producing Ability

(1) Production Ability in Stationary Culture

Culture media were prepared in which pretreated BDF-B and reagentglycerol, respectively, were added in place of glucose as a carbonsource in the LB culture medium, and called LB/BDF-B culture medium andLB/glycerol culture medium, respectively. The strain NEDO-01 (G.intermedius strain SIID9587), Gluconacetobacter xylinus strainATCC53582, Gluconacetobacter hansenii strain ATCC23769, andGluconacetobacter xylinus strain ATCC700178 (BPR2001) were eachinoculated on each of the LB/glycerol culture medium and the LB/BDF-Bculture medium and subjected to stationary culture at 30° C. for 7 daysto form a gelled film. The operation of adding a 1% (w/v) NaOH aqueoussolution thereto and performing autoclave treatment was repeated untilthe gelled film became white. Thereafter, the operation of adding waterand performing autoclave treatment was repeated until pH reached 7 orless for purification. The bacterial cellulose obtained by drying afterpurification was measured for the absolute dry weight. The results areshown in FIG. 12.

As shown in FIG. 12, G. hansenii strain ATCC23769 produced small weightsof bacterial celluloses in both of the LB/glycerol culture medium andthe LB/BDF-B culture medium. G. xylinus strain ATCC53582 and G. xylinusstrain ATCC700178 (BPR2001) produced relatively large weights ofbacterial celluloses in the LB/glycerol culture medium, whereas nobacterial cellulose production was observed in LB/BDF-B culture medium.In contrast, the strain NEDO-01 (G. intermedius strain SIID9587)produced comparably large weights of bacterial celluloses in both of theLB/glycerol culture medium and the LB/BDF-B culture medium. Theseresults showed that the strain NEDO-01 (G. intermedius strain SIID9587)could efficiently produce a bacterial cellulose by being subjected tostationary culture using either reagent glycerol or BDF-B as a carbonsource. Its feature of being capable of producing a bacterial celluloseusing BDF-B as a carbon source is a feature which other compared strainsdo not have, also advantageous on the practical side in which theby-product can be utilized, and greatly contributes to a reduction inproduction cost.

(2) Production Ability in Agitated Culture

The strains NEDO-01 (G. intermedius strain SIID9587), strain ATCC53582,and strain ATCC23769 were each inoculated on 10 mL of the HS culturemedium and subjected to stationary culture at 30° C. for 3 days forpre-preculture. Subsequently, the culture solution obtained by thepre-preculture was inoculated on 10 mL of the HS culture medium andsubjected to stationary culture at 30° C. for 3 days for preculture.Then, 100 mL of each of the main-culture medium with glycerol and themain culture medium with BDF-B of (1) of Example 2 was placed in abladed Erlenmeyer flask, and the preculture solution was inoculated inan amount corresponding to the same number of bacterial cells for eachbacterial strain thereon and subjected to shake culture for 3 days underconditions of 150 rpm and 30° C. for the main culture. Subsequently, abacterial cellulose in the main-culture solution was purified by themethod described in (1) of Example 2. However, shake was performed at60° C. and 80 rpm for 4 to 5 hours, followed by further shaking at 20°C. overnight. The purified bacterial cellulose was dried and measuredfor the absolute dry weight. The results are shown in FIG. 13.

As shown in FIG. 13, G. xylinus strain ATCC53582 was not observed toproduce a bacterial cellulose in each of the main-culture medium withglycerol and the main culture medium with BDF-B. For G. hansenii strainATCC23769, the absolute dry weight of the bacterial cellulose wasrelatively large when the main-culture medium with glycerol was used,but no bacterial cellulose production was observed when the main culturemedium with BDF-B was used. In contrast, for the strain NEDO-01 (G.intermedius strain SIID9587), the absolute dry weight of the bacterialcellulose was large when each of the main-culture medium with glyceroland the main culture medium with BDF-B was used. These results showedthat the strain NEDO-01 (G. intermedius strain SIID9587) couldefficiently produce the bacterial cellulose by either stationary cultureor agitated culture using either reagent glycerol or BDF-B as a carbonsource.

1. A bacterial cellulose produced by a bacterium, having a physicalcharacteristic of (a) below: (a) a transmittance of light at awavelength of 500 nm of water containing the bacterial cellulose at afinal concentration of 0.1±0.006% (w/w) of 35% or more.
 2. The bacterialcellulose according to claim 1, having a physical characteristic of (b)below: (b) a retention volume of a peak top of a chromatogram in gelpermeation chromatography performed under the following conditions i) tovi) of from 2.5 mL inclusive to 3.0 mL exclusive: i) column: a column6.0 mm in inside diameter and 15 cm in length, packed with amethacrylate polymer having a particle diameter of 9 μm; ii) guardcolumn: 4.6 mm in inside diameter and 3.5 cm in length; iii) columntemperature: 35° C.; iv) feed flow rate: 0.07 mL/minute; v) eluent: a 40to 42% (w/w) tetrabutylphosphonium hydroxide aqueous solution; and vi)final concentration of the bacterial cellulose in the eluent: 0.2%(w/w).
 3. The bacterial cellulose according to claim 1, produced by theassimilation of a glycerol-containing by-product generated in producinga biodiesel fuel from vegetable oil using the bacterium.
 4. Thebacterial cellulose according to claim 1, produced by the assimilationof one or two or more selected from the group consisting of sugar, asucrose-containing by-product generated in producing sugar, andhydrolysates thereof, and isomerized sugar, using the bacterium.
 5. Thebacterial cellulose according to claim 4, wherein the by-product ismolasses.
 6. The bacterial cellulose according to claim 1, wherein thebacterium is Gluconacetobacter intermedius.
 7. The bacterial celluloseaccording to claim 1, wherein the bacterium is Gluconacetobacterintermedius strain SIID9587 (accession number NITE BP-01495).
 8. Abacterium producing the bacterial cellulose according to claim
 1. 9.Gluconacetobacter intermedius strain SIID9587 (accession number NITEBP-01495) producing the bacterial cellulose according to claim 1.